Recombinant adeno-associated virus (rAAV) vectors have been developed for gene replacement therapy because they are non-pathogenic and exhibit a broad range of tissue specificity. These vectors generally retain the AAV inverted terminal repeats (ITRs) located at each end of a gene expression cassette, but lack the AAV rep and cap genes necessary for viral replication and packaging. Therefore rAAV cannot replicate, and viruses must be assembled in packaging cell lines with the rep and cap functions supplied in trans or expressed within the packaging cell itself. The gene expression cassette for insertion into the rAAV conventionally contains the therapeutic gene and the cis-regulatory elements including a promoter and a polyadenylation signal necessary for gene expression. In a conventional method of rAAV production, a gene expression cassette, located between the ITRs, is packaged in rAAV particles, which are then used in therapeutic applications.
The design and construction of the components, such as the plasmids and gene expression cassettes necessary for producing a recombinant AAV, can be quite labor intensive, due to the variety of plasmids and vectors available, and the need to modify the genes to fit into the appropriate plasmids. This complexity is further increased by pharmaceutical industry and governmental requirements that govern the process for obtaining approval of an rAAV for pharmaceutical use.